Method for in vitro molecular evolution of protein function

ABSTRACT

The present invention relates to a method for in vitro evolution of protein function. In particular, the method relates to the shuffling of nucleotide segments obtained from exonuclease digestion. The present inventors have shown that polynucleotide fragments derived from a parent polynucleotide sequence digested with an exonuclease can be combined to generate a polynucleofide sequence which encodes for a polypeptide having desired characteristics. This method may be usefully applied to the generation of new antibodies or parts thereof having modified characteristics as compared to the parent antibody.

This application claims priority under 35 U.S.C. §119(e) toPCT/GB98/01757 entitled “A method for in vitro evolution of proteinfunction” filed Jun. 16, 1998, which in turn claims priority from GBapplication 9712512.4 filed Jun. 16, 1997, the entire disclosure of eachbeing incorporated by reference herein.

FIELD OF THE INVENTION

The present invention relates to a method for in vitro molecularevolution of protein function, in particular by shuffling of DNAsegments obtained using an exonuclease.

BACKGROUND OF THE INVENTION

Protein function can be modified and improved in vitro by a variety ofmethods, including site directed mutagenesis (Alber et al, Nature, 5;330(6143):41-46, 1987) combinatorial cloning (Huse et al, Science,246:1275-1281, 1989; Marks et al, Biotechnology, 10: 779-783, 1992) andrandom mutagenesis combined with appropriate selection systems (Barbaset al, PNAS. USA, 89: 4457-4461, 1992).

The method of random mutagenesis together with selection has been usedin a number of cases to improve protein function and two differentstrategies exist. Firstly, randomisation of the entire gene sequence incombination with the selection of a variant (mutant) protein with thedesired characteristics, followed by a new round of random mutagenesisand selection. This method can then be repeated until a protein variantis found which is considered optimal (Schier R. et al, J. Mol. Biol.1996 263 (4): 551-567). Here, the traditional route to introducemutations is by error prone PCR (Leung et al, Technique, 1: 11-15, 1989)with a mutation rate of ≈0.7%. Secondly, defined regions of the gene canbe mutagenized with degenerate primers, which allows for mutation ratesup to 100% (Griffiths et al, EMBO. J, 13: 3245-3260, 1994; Yang et al,J. Mol. Biol. 254: 392-403, 1995). The higher the mutation rate used,the more limited the region of the gene that can be subjected tomutations.

Random mutation has been used extensively in the field of antibodyengineering. In vivo formed antibody genes can be cloned in vitro(Larrick et al, Biochem. Biophys. Res. Commun. 160: 1250-1256, 1989) andrandom combinations of the genes encoding the variable heavy and lightgenes can be subjected to selection (Marks et al, Biotechnology, 10:779-783, 1992). Functional antibody fragments selected can be furtherimproved using random mutagenesis and additional rounds of selections(Schier R. et al, J. Mol. Biol. 1996 263 (4): 551-567) .

The strategy of random mutagenesis is followed by selection. Variantswith interesting characteristics can be selected and the mutated DNAregions from different variants, each with interesting characteristics,are combined into one coding sequence (Yang et al, J. Mol. Biol. 254:392-403, 1995). This is a multi-step sequential process, and potentialsynergistic effects of different mutations in different regions can belost, since they are not subjected to selection in combination. Thus,these two strategies do not include simultaneous mutagenesis of definedregions and selection of a combination of these regions. Another processinvolves combinatorial pairing of genes which can be used to improve egantibody affinity (Marks et al, Biotechnology, 10: 779-783, 1992). Here,the three CDR-regions in each variable gene are fixed and thistechnology does not allow for shuffling of individual gene segments inthe gene for the variable domain, for example, including the CDRregions, between clones.

The concept of DNA shuffling (Stemmer, Nature 370: 389-391, 1994)utilizes random fragmentation of DNA and assembly of fragments into afunctional coding sequence. In this process it is possible to introducechemically synthesized DNA sequences and in this way target variation todefined places in the gene which DNA sequence is known (Crameri et al,Biotechniques, 18: 194-196, 1995). In theory, it is also possible toshuffle DNA between any clones. However, if the resulting shuffled geneis to be functional with respect to expression and activity, the clonesto be shuffled have to be related or even identical with the exceptionof a low level of random mutations. DNA shuffling between geneticallydifferent clones will generally produce non-functional genes.

Selection of functional proteins from molecular libraries has beenrevolutionized by the development of the phage display technology(Parmley et al, Gene, 73: 305-391 1988; McCafferty et al, Nature, 348:552-554, 1990; Barbas et al, PNAS. USA, 88: 7978-7982, 1991). Here, thephenotype (protein) is directly linked to its corresponding genotype(DNA) and this allows for directly cloning of the genetic material whichcan then be subjected to further modifications in order to improveprotein function. Phage display has been used to clone functionalbinders from a variety of molecular libraries with up to 10¹¹transformants in size (Griffiths et al, EMBO. J. 13: 3245-3260, 1994).Thus, phage display can be used to directly clone functional bindersfrom molecular libraries, and can also be used to improve further theclones originally selected.

Random combination of DNA from different mutated clones in combinationwith selection of desired function is a more efficient way to searchthrough sequence space as compared to sequential selection andcombination of selected clones.

SUMMARY OF THE INVENTION

According to one aspect of the present invention, there is provided amethod for generating a polynucleotide sequence or population ofsequences from a parent polynucleotide sequence encoding one or moreprotein motifs, comprising the steps of

a) digesting the parent polynucleotide sequence with an exonuclease togenerate a population of fragments;

b) contacting said fragments with a template polynucleotide sequenceunder annealing conditions;

c) amplifying the fragments that anneal to the template in step b) togenerate at least one polynucleotide sequence encoding one or moreprotein motifs having altered characteristics as compared to the one ormore protein motifs encoded by said parent polynucleotide.

The parent polynucleotide is preferably double-stranded and the methodfurther comprises the step of generating single-stranded polynucleotidesequence from said double-stranded fragments prior to step b). Further,the template polynucleotide is preferably the parent polynucleotidesequence or at least a polynucleotide sequence having sequence in commonwith the parent nucleotide sequence so that the fragments will hybridizewith the template under annealing conditions. For example, if the parentpolynucleotide is an antibody, the template may be a different antibodyhaving constant domains or framework regions in common.

Therefore, typically, there is provided a method of combiningpolynucleotide fragments to generate a polynucleotide sequence orpopulation of sequences of desired characteristics, which methodcomprises the steps of:

(a) digesting a linear parent double-stranded polynucleotide encodingone or more protein motifs with an exonuclease to generate a populationof double stranded fragments of varying lengths;

(b) obtaining single-stranded polynucleotides from said double-strandedfragments; and

(c) assembling a polynucleotide sequence from the sequences derived fromstep (b).

Preferably the method further comprises the step of (d) expressing theresulting protein encoded by the assembled polynucleotide sequence andscreening the protein for desired characteristics.

Prior to assembling the polynucleotide sequence in step (c) the doublestranded sequences are preferably purified and then mixed in order tofacilitate assembly. By controlling the reaction time of the exonucleasethe size of the polynucleotide fragments may be determined. Determiningthe lengths of the polynucleotide fragments in this way avoids thenecessity of having to provide a further step such as purifying thefragments of desired length from a gel.

Further, as some exonuclease digests polynucleotide sequences from boththe 3′ and the 5′ ends, the fragments selected may center around themiddle of the gene sequence if this particular region of sequence isdesired. This sequence from the middle of a gene may be mutated randomlyby, for example, error prone PCR and desirable for the shufflingprocess.

However, in some cases it may be desirable not to shuffle the sequencefrom the middle of the gene. This may be prevented by choosing longfragments after exonuclease treatment. Conversely, if it is desirable toshuffle the middle of the gene sequence short exonuclease treatedfragments may be used.

In order to generate a polynucleotide sequence of desiredcharacteristics the parent double-stranded polynucleotide encoding oneor more protein motifs may be subjected to mutagenesis to create aplurality of differently mutated derivatives thereof. Likewise, a parentdouble-stranded polynucleotide may be obtained already encoding aplurality of variant protein motifs of unknown sequence.

Random mutation can be accomplished by any conventional method asdescribed above, but a suitable method is error-prone PCR.

It is preferable to use PCR technology to assemble the single-strandedpolynucleotide fragments into the double-stranded polynucleotidesequence.

The polynucleotide sequence is preferably DNA although RNA may be used.For simplicity the term polynucleotide will now be used in the followingtext in relation to DNA but it will be appreciated that the presentinvention is applicable to both RNA and DNA.

Any exonuclease that digests polynucleotide from the 3′ prime end to the5′ prime end or from both the 3′ and the 5′ end may be used. Examples ofa suitable exonuclease which may be used in accordance with the presentinvention include BAL31 and Exonuclease III.

BAL31 is a exonuclease that digests and removes nucleotide bases fromboth the 3′ and the 5′ ends of a linear polynucleotide molecule. Theenzyme uses Ca2+ as a co-factor which can be bound in complex with EGTA(Ethylene Glycol bis(β-amino ethyl Ether) N,N,N′,N′-tetra acetic acid).EGTA does not bind Mg2+ which is necessary for the subsequent PCRprocess. Linear DNA sequences are digested with BAL31 and the reactionstopped at different time points by the addition of EGTA. The individualdigested fragments are purified, mixed and reassembled with PCRtechnology. The assembled (reconstituted) gene may then be cloned intoan expression vector for expressing the protein. The protein may then beanalyzed for improved characteristics.

The PCR technique uses a template, which may be the wild type sequenceor a reconstituted sequence in accordance with the present invention.The fragments hybridize with the template at the appropriate regions(i.e. where the homology between the two strands is at its highest) andthe remaining sequence is generated by elongation of the fragment usingthe template in accordance with the PCR technique.

The method of the present invention provides several advantages overknown shuffling techniques. For example, in other DNA shufflingtechniques the process itself introduces mutations over the entire genesequence. The present invention allows for the concentration ofmutations on i) the flanking regions after recombination of wild typefragments on either an already recombined template created by the methodof the present invention, a template mutated in any other way or a gene(or gene combination, for example, a combination of antibody genes)having a desired sequence; or ii) the middle region after recombinationof mutated fragments created by the method of the present invention on awild type template.

In other words, if it is desirable to provide a gene having mutationsconcentrated in its flanking regions, a wild type fragment relating tothe middle region of the gene may be used in conjunction with areconstituted and/or mutated template sequence for the PCR process. Inthis way, the PCR process generates complementary sequence to thereconstituted/mutated template sequence as it elongates the wild typefragment. Therefore, the resulting sequence will have substantially amiddle region corresponding to the wild type sequence and flankingregions with incorporated mutations.

Conversely, if it is desirable to provide a gene having mutationsconcentrated in its middle region, a reconstituted and or mutatedfragment corresponding to the middle region of the gene may be used inconjunction with a wild type template in the PCR process. In this way,the PCR process, by elongating the mutated fragment using the wild typetemplate, generates a sequence having substantially a mutated middleregion and wild type flanking regions.

Further, the method of the present invention produces a set ofprogressively shortened DNA fragments for each time point a DNA sampleis taken from the BAL31 treatment. The DNA samples may be collected andpooled together or, optionally, individual samples may be chosen andused in the method. Thus the present invention allows a selection ofwhat DNA samples are to be used in the recombination system and therebyoffers a further degree of control.

The method of the present invention may be carried out on anypolynucleotide which codes for a particular product for example anyprotein having binding or catalytical properties e.g. antibodies orparts of antibodies, enzymes or receptors. Further, any polynucleotidethat has a function that may be altered for example catalytical RNA maybe shuffled in accordance with the present invention. It is preferablethat the parent polynucleotide encoding one or more protein motif is atleast 12 nucleotides in length, more preferably at least 20 nucleotidesin length, even more preferably more than 50 nucleotides in length.Polynucleotides being at least 100 nucleotides in length or even atleast 200 nucleotides in length may be used. Where parentpolynucleotides are used that encoded for large proteins such as enzymesor antibodies, these may be many hundreds or thousands of bases inlength. The present invention may be carried out on any size of parentpolynucleotide.

The present invention also provides polynucleotide sequences generatedby the method described above having desired characteristics. Thesesequences may be used for generating gene therapy vectors andreplication-defective gene therapy constructs or vaccination vectors forDNA-based vaccinations. Further, the polynucleotide sequences may beused as research tools.

The present invention also provides a polynucleotide library ofsequences generated by the method described above from which apolynucleotide may be selected which encodes a protein having thedesired characteristics. It is preferable that the polynucleotidelibrary is a DNA or cDNA library.

The present inventions also provides proteins such as antibodies,enzymes, and receptors having characteristics different to that of thewild type produced by the method described above. These proteins may beused individually or within a pharmaceutically acceptable carrier asvaccines or medicaments for therapy, for example, as immunogens,antigens or otherwise in obtaining specific antibodies. They may also beused as research tools.

The desired characteristics of a polynucleotide generated by the presentinvention or a protein encoded by a polynucleotide generated by thepresent invention may be any variation in the normal activity of thewild type (parent) polynucleotide or the polypeptide, protein or proteinmotifs it encodes. For example, it may be desirable to reduce orincrease the catalytic activity of an enzyme, or improve or reduce thebinding specificity of an antibody. Further, if the protein, orpolynucleotide is an immunogen, it may be desirable to reduce orincrease its ability to obtain specific antibodies against it. Theparent polynucleotide preferably encodes one or more protein motifs.These are defined by regions of polynucleotide sequence that encodepolypeptide sequence having or potentially having characteristic proteinfunction. For example, a protein motif may define a portion of a wholeprotein, i.e. an epitope or a cleavage site or a catalytic site etc.However, within the scope of the present invention, an expressed proteinmotif does not have to display activity, or be “correctly” folded.

It may be desirable to modify a protein so as to alter the conformationof certain epitopes, thereby improving its antigenicity and/or reducingcross-reactivity. For example, should such a protein be used as anantigen, the modification may reduce any cross-reaction of raisedantibodies with similar proteins.

Although the term “enzyme” is used, this is to interpreted as alsoincluding any polypeptide having enzyme-like activity, i.e. a catalyticfunction. For example, polypeptides being part of an enzyme may stillpossess catalytic function. Likewise, the term “antibody” should beconstrued as covering any binding substance having a binding domain withthe required specificity. This includes antibody fragments, derivatives,functional equivalents and homologues of antibodies, including syntheticmolecules and molecules whose shape mimics that of an antibody enablingit to bind an antigen or epitope. Examples of antibody fragments,capable of binding an antigen or other binding partner are Fab fragmentconsisting of the VL, VH, Cl and CH1 domains, the Fd fragment consistingof the VH and CH1 domains; the Fv fragment consisting of the VL and VHdomains of a single arm of an antibody; the dAb fragment which consistsof a VH domain; isolated CDR regions and F(ab′)2 fragments, a bivalentfragment including two Fab fragments linked by a disulphide bridge atthe hinge region. Single chain Fv fragments are also included.

In order to obtain expression of the generated polynucleotide sequence,the sequence may be incorporated in a vector having control sequencesoperably linked to the polynucleotide sequence to control itsexpression. The vectors may include other sequences such as promoters orenhancers to drive the expression of the inserted polynucleotidesequence, further polynucleotide sequences so that the protein encodedfor by the polynucleotide is produced as a fusion and/or nucleic acidencoding secretion signals so that the protein produced in the host cellis secreted from the cell. The protein encoded for by the polynucleotidesequence can then be obtained by transforming the vectors into hostcells in which the vector is functional, culturing the host cells sothat the protein is produced and recovering the protein from the hostcells or the surrounding medium. Prokaryotic and eukaryotic cells areused for this purpose in the art, including strains of E. coli, yeast,and eukaryotic cells such as COS or CHO cells. The choice of host cellcan be used to control the properties of the protein expressed in thosecells, e.g. controlling where the protein is deposited in the host cellsor affecting properties such as its glycosylation.

The protein encoded by the polynucleotide sequence may be expressed bymethods well known in the art. Conveniently, expression may be achievedby growing a host cell in culture, containing such a vector, underappropriate conditions which cause or allow expression of the protein.

Systems for cloning and expression of a protein in a variety ofdifferent host cells are well known. Suitable host cells includebacteria, eukaryotic cells such as mammalian and yeast, and baculovirussystems. Mammalian cell lines available in the art for expression of aheterologous polypeptide include Chinese hamster ovary cells, HeLacells, baby hamster kidney cells, COS cells and many others. A common,preferred bacterial host is E. coli.

Suitable vectors can be chosen or constructed, containing appropriateregulatory sequences, including promoter sequences, terminatorfragments, polyadenylation sequences, enhancer sequences, marker genesand other sequences as appropriate. Vectors may be plasmids, viral e.g.‘phage, or phagemid, as appropriate. For further details see, forexample, Molecular Cloning: a Laboratory Manual: 2nd edition, Sambrooket al., 1989, Cold Spring Harbor Laboratory Press. Many known techniquesand protocols for manipulation of polynucleotide sequences, for examplein preparation of polynucleotide constructs, mutagenesis, sequencing,introduction of DNA into cells and gene expression, and analysis ofproteins, are described in detail in Current Protocols in MolecularBiology, Ausubel et al. eds., John Wiley & Sons, 1992.

The FIND system can be used for the creation of DNA libraries comprisingvariable sequences which can be screened for the desired proteinfunction in a number of ways. Phage display may be used for selectingbinding (Griffith et al., EMBO J. 113: 3245-3260, 1994); screening forenzyme function (Crameri A. et al, Nature 1998 15; 391 (6664):288-291;Zhang J. H. et al, PNAS. USA 1997 94 (9): 4504-4509; Warren M. S. et al,Biochemistry 1996, 9; 35(27): 8855-8862).

A protein provided by the present invention may be used in screening formolecules which affect or modulate its activity or function. Suchmolecules may be useful in a therapeutic (possibly includingprophylactic) context.

The present invention also provides vectors comprising polynucleotidesequences generated by the method described above.

The present inventions also provides compositions comprising eitherpolynucleotide sequences, vectors comprising the polynucleotidesequences or proteins generated by the method described above and apharmaceutically acceptable carrier or a carrier suitable for researchpurposes.

The present invention also provides a method comprising, following theidentification of the polynucleotide or polypeptide having desiredcharacteristics by the method described above, the manufacture of thatpolypeptide or polynucleotide in whole or in part, optionally inconjunction with additional polypeptides or polynucleotides.

Following the identification of a polynucleotide or polypeptide havingdesired characteristics, these can then be manufactured to providegreater numbers by well known techniques such as PCR, cloning aexpression within a host cell. The resulting polypeptides orpolynucleotides may be used in the preparation of medicaments fordiagnostic use, pharmaceutical use, therapy etc. This is discussedfurther below. Alternatively, the manufactured polynucleotide,polypeptide may be used as a research tool, i.e. antibodies may be usedin immunoassays, polynucleotides may be used a hybridization probes orprimers.

The polypeptides or polynucleotides generated by the method of theinvention and identified as having desirable characteristics can beformulated in pharmaceutical compositions. These compositions maycomprise, in addition to one of the above substances, a pharmaceuticallyacceptable excipient, carrier, buffer, stabilizer or other materialswell known to those skilled in the art. Such materials should benon-toxic and should not interfere with the efficacy of the activeingredient. The precise nature of the carrier or other material maydepend on the route of administration, e.g. oral, intravenous, cutaneousor subcutaneous, nasal, intramuscular, intraperitoneal routes.

Pharmaceutical compositions for oral administration may be in tablet,capsule, powder or liquid form. A tablet may include a solid carriersuch as gelatin or an adjuvant. Liquid pharmaceutical compositionsgenerally include a liquid carrier such as water, petroleum, animal orvegetable oils, mineral oil or synthetic oil. Physiological salinesolution, dextrose or other saccharide solution or glycols such asethylene glycol, propylene glycol or polyethylene glycol may beincluded.

For intravenous, cutaneous or subcutaneous injection, or injection atthe site of affliction, the active ingredient will be in the form of aparenterally acceptable aqueous solution which is pyrogen-free and hassuitable pH, isotonicity and stability. Those of relevant skill in theart are well able to prepare suitable solutions using, for example,isotonic vehicles such as Sodium Chloride Injection, Ringer's Injection,Lactated Ringer's Injection. Preservatives, stabilizers, buffers,antioxidants and/or other additives may be included, as required.

Whether it is a polypeptide, e.g. an antibody or fragment thereof, anenzyme, a polynucleotide or nucleic acid molecule, identified followinggeneration by the present invention that is to be given to anindividual, administration is preferably in a “prophylacticallyeffective amount” or a “therapeutically effective amount” (as the casemay be, although prophylaxis may be considered therapy), this beingsufficient to show benefit to the individual. The actual amountadministered, and rate and time-course of administration, will depend onthe nature and severity of what is being treated. Prescription oftreatment, e.g. decisions on dosage etc, is within the responsibility ofgeneral practitioners and other medical doctors, and typically takesaccount of the disorder to be treated, the condition of the individualpatient, the site of delivery, the method of administration and otherfactors known to practitioners. Examples of the techniques and protocolsmentioned above can be found in Remington's Pharmaceutical Sciences,16th edition, Osol, A. (ed), 1980.

Alternatively, targeting therapies may be used to deliver the activeagent more specifically to certain types of cell, by the use oftargeting systems such as antibody or cell specific ligands. Targetingmay be desirable for a variety of reasons; for example if the agent isunacceptably toxic, or if it would otherwise require too high a dosage,or if it would not otherwise be able to enter the target cells.

Instead of administering these agents directly, they could be producedin the target cells by expression from an encoding gene introduced intothe cells, e.g. in a viral vector (a variant of the VDEPT technique).The vector could be targeted to the specific cells to be treated, or itcould contain regulatory elements which are switched on more or lessselectively by the target cells.

Alternatively, the agent could be administered in a precursor form, forconversion to the active form by an activating agent produced in, ortargeted to, the cells to be treated. This type of approach is sometimesknown as ADEPT or VDEPT; the former involving targeting the activatingagent to the cells by conjugation to a cell-specific antibody, while thelatter involves producing the activating agent, e.g. an enzyme, in avector by expression from encoding DNA in a viral vector (see forexample, EP-A-415731 and WO 90/07936).

A composition may be administered alone or in combination with othertreatments, either simultaneously or sequentially dependent upon thecondition to be treated.

As a further alternative, the polynucleotide identified as havingdesirable characteristics following generation by the method of thepresent invention could be used in a method of gene therapy, to treat apatient who is unable to synthesize the active polypeptide encoded bythe polynucleotide or unable to synthesize it at the normal level,thereby providing the effect provided by the corresponding wild-typeprotein.

Vectors such as viral vectors have been used in the prior art tointroduce polynucleotides into a wide variety of different target cells.Typically the vectors are exposed to the target cells so thattransfection can take place in a sufficient proportion of the cells toprovide a useful therapeutic or prophylactic effect from the expressionof the desired polypeptide. The transfected nucleic acid may bepermanently incorporated into the genome of each of the targeted tumourcells, providing long lasting effect, or alternatively the treatment mayhave to be repeated periodically.

A variety of vectors, both viral vectors and plasmid vectors, are knownin the art, see U.S. Pat. No. 5,252,479 and WO 93/07282. In particular,a number of viruses have been used as gene transfer vectors, includingpapovaviruses, such as SV40, vaccinia virus, herpes viruses, includingHSV and EBV, and retroviruses. Many gene therapy protocols in the priorart have used disabled murine retroviruses.

As an alternative to the use of viral vectors other known methods ofintroducing nucleic acid into cells includes electroporation, calciumphosphate co-precipitation, mechanical techniques such asmicroinjection, transfer mediated by liposomes and direct DNA uptake andreceptor-mediated DNA transfer.

As mentioned above, the aim of gene therapy using nucleic acid encodinga polypeptide, or an active portion thereof, is to increase the amountof the expression product of the nucleic acid in cells in which thelevel of the wild-type polypeptide is absent or present only at reducedlevels. Such treatment may be therapeutic in the treatment of cellswhich are already cancerous or prophylactic in the treatment ofindividuals known through screening to have a susceptibility allele andhence a predisposition to, for example, cancer.

The present invention also provides a kit for generating apolynucleotide sequence or population of sequences of desiredcharacteristics comprising an exonuclease and components for carryingout a PCR technique, for example, thermostable DNA (nucleotides) and astopping device, for example, EGTA.

The present applicants have termed the technology described above asFIND (Fragment Induced Nucleotide Diversity).

As outlined above, the FIND programme, in accordance with the presentinvention conveniently provides for the creation of mutated antibodygene sequences and their random combination to functional antibodieshaving desirable characteristics. As an example of this aspect of theinvention, the antibody genes are mutated by error prone PCR whichresults in a mutation rate of approximately 0.7%. The resulting pool ofmutated antibody genes are then digested with an exonuclease, preferablyBAL31, and the reaction inhibited by the addition of EGTA at differenttime points, resulting in a set of DNA fragments of different sizes.These may then be subjected to PCR based reassembly as described above.The resulting reassembled DNA fragments are then cloned and a genelibrary constructed. Clones may then be selected from this library andsequenced.

A further application of the FIND technology is the generation of apopulation of variable DNA sequences which can be used for furtherselections and analyses. Besides encoding larger proteins, e.g. antibodyfragments and enzymes, the DNA may encode peptides where the moleculesfunctional characteristics can be used for the design of differentselection systems. Selection of recombined DNA sequences encodingpeptides has previously been described (Fisch et al PNAS. USA Jul. 23,1996; 93 (15): 7761-7766). In addition, the variable DNA population canbe used to produce a population of RNA molecules with e.g. catalyticactivities. Vaish et al (PNAS. USA Mar. 3, 1998; 95 (5): 2158-2162)demonstrated the design of functional systems for the selection ofcatalytic RNA and Eckstein F (Ciba Found. Symp. 1997; 209; 207-212) hasoutlined the applications of catalytic RNA by the specific introductionof catalytic RNA in cells. The FIND system may be used to further searchthrough the sequence space in the selection of functionalpeptides/molecules with catalytic activities based on recombined DNAsequences.

Aspects and embodiments of the present invention will now beillustrated, by way of example, with reference to the accompanyingfigures. Further aspects and embodiments will be apparent to thoseskilled in the art. All documents mentioned in this text areincorporated herein by reference.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the principle steps in the shuffling of specific DNAsequences between different clones;

FIG. 2 shows the principle steps in the PCR elongation of exonucleasetreated gene sequences;

FIG. 3 shows the principle steps in the PCR elongation of long fragmentsof exonuclease treated gene sequences. The use of long fragments resultsin the middle region of the gene not being recombined. This region mayhowever contain random mutations and the middle of the gene sequence maythus differ form other clones. The middle region of the sequence maydiffer in length, but by using longer primers the middle region may becovered;

FIG. 4 shows the principle steps in the PCR elongation of shortfragments of exonuclease treated gene sequences. The use of shortfragments results in the middle region of the gene being recombined. Ifa longer reaction time is used for the exonuclease digestion a set offragments of differing lengths are produced. If the fragments are short,some fragments will be located away from the middle region of the genesequence thereby allowing recombination of the middle sequence;

FIG. 5 shows the appearance of DNA at different fixed time intervalsafter digestion with BAL31 Nuclease. The DNA was mixed with the enzymeand incubated at 30° C. At different time points samples were removedand the enzymatic activity stopped by addition of 20 mM EGTA. Thesamples from the different time points were purified and analyzed on a2% agarose gel. The samples are indicated as follows: 1 Kb=DNA molecularmarker 1; 2-10 m=2 to 10 minutes BAL31 incubation samples;

FIG. 6 shows A) the theoretical insert after restriction digestion ofthe fragment resulting from the primer combination FIND 1, pBR322NheI-forward —STOP—primer with pBR322-EagI-reversed-primer. This istermed FIND 1 and SEQ ID #5; and B) the theoretical insert afterrestriction digestion of the fragment resulting from the primercombination pBR322 HindIII forward primer and pBR322 SalI reverse stopprimer. This is termed FIND 3 and SEQ ID #6; and

FIG. 7 shows the experimentally determined sequences of the 2 first FINDclones after automated sequencing. A) shows FIND 1 sequence with theSTOP codon marked in bold (SEQ ID #7); and B) shows the FIND 3 sequencewith the STOP codon shown in underline text (SEQ ID #8).

FIG. 8 shows the sequence of pEXmide V (4055 bp) NcoI- and Sal I-sitesare marked in underlined text (SEQ ID #9).

DETAILED DESCRIPTION AND EXEMPLIFICATION OF THE INVENTION

One aspect of the DNA shuffling procedure can be illustrated by thesteps shown in FIG. 1. The gene encoding the tetracycline-resistance(Tet-R) in the plasmid. pBR322 is used in this example. Two clones weregenerated by site directed mutagenesis: one with an engineered stopcodon close to the 5′ terminus and one with a stop codon close to the 3′terminus of the Tet-R gene. The phenotype of these two genes istetracycline sensitive. By mixing the two clones in equimolar amountsand digesting with BAL31 revertants were selected. After cloning thereassembled genes (with combination between the two genes carrying thetwo stop codons) revertants with a frequency of 16% were detected, i.e.16% of the clones were tetracycline resistant. The experiment used theampicillin-resistance in pBR322 for primary selection and thenindividual Amp-R clones were tested under tetracycline selection (seethe overview in FIG. 1 and the theoretical view in FIG. 2).

A more detailed description of examples of the present invention aregiven below.

Reagents

AmpliTaq® polymerase was purchased from Perkin-Elmer Corp., dNTPs fromBoehringer Mannheim Biochemica (Mannheim, Germany), and BAL31 Nucleasefrom New England Biolabs Inc. (Beverly, USA). Klenow enzyme waspurchased from Amersham. All restriction enzymes were purchased fromBoehringer Mannheim Biochemica (Mannheim, Germany). Ethidium bromide waspurchased from Bio-Rad Laboratories (Bio-Rad Laboratories, Hercules,Calif., USA). T4 DNA Ligase was purchased from Appligene Inc.(Pleasanton, Calif., USA).

All primers were designed in the laboratory and synthesized with anApplied Biosystems 391 DNA-synthesiser.

PCR

All Polymerase Chain Reactions (PCR) were carried out in a automaticthermocycler (Perkin-Elmer Cetus 480, Norwalk, Conn., USA). PCRtechniques for the amplification of nucleic acid are described in U.S.Pat. No. 4,683,195. The PCR reactions were run at varying amounts ofcycles consisting of following profile: denaturation (94° C., 1 minute),primer annealing (55° C., 1 minute) and extension (72° C., 1 minute)using a 1 second ramp time. The PCR reactions contained, unlessotherwise noted, 5 μl of each primer (20 μM), 8 μl of dNTP (1.25 mM eachof dTTP, DATP, dCTP and dGTP), 10 μl 10×reaction buffer, 0.5 μlAmpliTaq® thermostable DNA polymerase (5 U/μl) (Perkin-Elmer Corp.), andwater to a final volume of 100 μl. In all PCR experiments theseparameters were used and the number of reaction cycles was varied.References for the general use of PCR techniques include Mullis et al,Cold Spring Harbor Symp. Quant. Biol., 51:263, (1987), Ehrlich (ed), PCRtechnology, Stockton Press, New York, 1989, Ehrlich et al, Science,252:1643-1650, (1991), “PCR protocols; A Guide to Methods andApplications”, Eds. Innis et al, Academic Press, New York, (1990).

Sequencing

All constructs have been sequenced by the use of a Taq Dyedeoxy™Terminator Cycle Sequencing Kit. The sequencing was performed on a ABIPrism 373 DNA Sequencer.

Agarose Electrophoresis

Agarose electrophoresis of DNA was performed with 2% agarose gelscomposed of 1% NuSieve® GTG® Low Melting AGAROSE (FMC Bioproducts,Rockland, Me., USA) and 1% AMRESCO® Agarose (AMRESCO, SOLON, Ohio, USA)with 0.25 μg/ml ethidium bromide in Tris-acetate buffer (TAE-buffer0.04M Tris-acetate, 0.001M EDTA) . Samples for electrophoresis weremixed with a sterile filtrated loading buffer composed of 25% Ficoll andBromphenolic blue and loaded into wells in a the 2% agarose gel. Theelectrophoresis was run at 90 V for 45 minutes unless otherwise statedin Tris-acetate buffer with 0.25 μg/ml ethidium bromide. Bands ofappropriate size were gel-purified using the Qiaquick Gel Extraction Kit(Qiagen GmbH, Hilden, Germany). As molecular weight standard, DNAmolecular weight marker 1 (Boehringer Mannheim GmbH, Germany) was used.The DNA-concentration of the gel extracted products were estimated usinga spectrophotometer (see FIG. 5).

Bacterial Strains

The Escherichia coli-strain E.coli BMH71-18 (supE thi Δ(lac-proAB) F′[proAB⁺ lacI^(q) Δ(lacZ)M15]), was used as a bacterial host fortransformations. Chemically competent cells of this strain were producedbasically as described Hanahan, D. 1983. Studies on transformation ofEscherichia coli with plasmids. J. Mol. Biol. 166: 557-580.Electrocompetent cells of this bacterial strain were produced (Dower, W.J., J. F. Miller, and C. W. Ragsdale. 1988: High efficiencytransformation of E.coli by high voltage electroporation. Nucleic AcidsRes. 16:6127).

Plasmids

The tetracycline resistance-gene of pBR322 is 1191 bp (basepairs) long.A deleted tetracycline resistance-gene variant of plasmid pBR322 wasconstructed by cleaving the plasmid with the restriction enzymes SalIand BamHI. This resulted in removal of a 276 bp fragment inside thetetracycline gene. A cleavage reaction with HindIII and EagI and thedeleted plasmid would theoretically lead to a 634 bp cleavage-product,whereas a wildtype pBR322 cleaved with these enzymes produces a 910 bpproduct. The resulting protruding single stranded overhangs on thedeleted plasmid after cleavage were treated with Klenow enzyme togenerate double-stranded ends at both ends of the plasmid. These endswere then blunt-end ligated according to Molecular cloning; A LABORATORYMANUAL (Second Edition, Cold Spring Harbor Laboratory Press, 1989). Theresulting plasmid was transformed into chemically competent E.coliBMH71-18 and plated onto ampillicin-containing plates (100 μg/ml). Whenreplated onto tetracycline-containing agarplates (10 μg/ml) the colonieswere tetracycline sensitive.

External Primers

Two external primers surrounding the tetracycline gene of pBR322 weredesigned with the following sequences including designated uniquerestriction sites:

pBR322 HindIII forward primer:

5′-CAGCTTATCATCGATAAGCTTTAATGCGGTAGTTTAT-3′ (SEQ ID #1)

and pBR322-EagI-reversed-primer:

5′-CGTAGCCCAGCGCGTCGGCCGCCATGCCGGCGATAATG-3′ (SEQ ID #2)

To show that the two external primers covers the functional parts of thetetracycline-gene, a PCR reaction with the above mentioned profile wasused for a 30 cycles-PCR with pBR322 (250 ng) as a template and theexternal primers described above. This yielded a PCR-product of 910 bpafter subsequent cleavage with HindIII and EagI. When this restrictionproduct was cloned in a likewise restriction-digested pBR322 plasmid,the plasmid encoded a tetracycline resistant phenotype. This wasdetected after transformation of a ligation of plasmid and 910 bpPCR-product into E.coli host BMH 7118 plated on tetracycline containingagar-plates (10 μg/ml).

STOP-containing Primers

Two pBR322 forward mutagenic primers and two pBR322 reversed primerscontaining unique restriction-sites and one STOP codon each at varioussites were constructed. These were:

pBR322 NheI forward STOP:

5′-CACTATGGCGTGCTGCTAGCGCTATATGCGTTGATGCAATTTCTATGAGCACCCGTTCT-3′. (SEQID #3)

pBR322 SalI reversed STOP:

5′-TCTCAAGGGCATCGGTCGACGCTCTCCCTTATGCGACTCCTGCATTAGGAATCAGCCCAGTAGTA-3′(SEQ ID #4)

Generation of STOP-codon Containing Variants of pBR322 Plasmids

Four different variants of the tetracycline gene were constructed. Acombination of one mutated forward or reversed primer with thecorresponding external forward or reversed primer was used inPCR-reactions to generate mutated inserts. Plasmid pBR322 was used as atemplate (250 ng) in 40 PCR-cycles. The resulting restriction digestedfragments were then cloned into tetracycline deleted pBR322, and theresulting clones were called FIND 1 and FIND 3.

The following primer combinations were used: FIND 1, pBR322NheI-forward-STOP-primer with pBR322-EagI-reversed-primer. Thiscombination gave the insert after restriction digestion as shown in FIG.6A; and FIND 3, pBR322 HindIII forward primer and pBR322 SalI reversedSTOP primer. This combination gave the insert after restrictiondigestion as shown in FIG. 6B.

The amplified PCR-products were analysed on a 2% agarose gel. Theelectrophoresis was run at 90V for 40 minutes as described above. Bandsof appropriate size (1000 bp), as compared to the molecular weightstandard, were cut out and gel-purified using the Qiaquick GelExtraction Kit. The four different STOP-containing inserts were thencleaved with the restriction-enzymes designated in the primers above.For each insert a pool of plasmid pBR322 was cleaved with the sameenzymes, and these four combinations were then ligated and transformedinto chemically competent E coli BMH 71-18 according to the modifiedprotocol of Detlef (Modified Hanahan, revised M. Scott, F. Hochstenbachand D. Güssow 1989). The transformants were plated onto ampicillincontaining agar-plates (50 μg/ml). When replated on tetracyclinecontaining agar-plates (10 μg/ml) no colonies survived, confirming thefunctional effect of the introduced STOP-codon in the tetracycline-gene.Plasmids of the four different FIND-clones were prepared with QiagenPlasmid Midi Kit (Qiagen Inc., Chatsworth, Calif., USA). The plasmids ofthe four clones were sequenced by the use of a Taq Dyedeoxy™ TerminatorCycle Sequencing Kit. The sequencing was performed on a ABI Prism 373DNA Sequencer. The STOP-codons were confirmed and the inserts to becorrect.

FIND Experiment I

Generation of FIND-fragments for BAL31 Nuclease Digestion

PCR-fragment of FIND 1 and FIND 3 were generated by runningPCR-reactions with FIND 1 and FIND 3-plasmids as templates (500 ng) andwith the two external primers, pBR322 HindIII forward primer andpBR322-EagI-reversed-primer. PCR-cycles were as described above for 30cycles. The amplified PCR-products were mixed with 20 μl of loadingbuffer (25% Ficoll and Bromphenolic blue) and analysed on a 2% agarosegel. The electrophoresis was run at 90V for 35 minutes as previouslydescribed. Bands of appropriate size were cut out and gel-purified usingthe Qiaquick Gel Extraction Kit. The DNA-concentration was estimated to112.25 μg/ml for the FIND-1 PCR-fragment and to 110 μg/ml for the FIND-3PCR-fragment.

BAL31 Nuclease Treatment

5 μg each of FIND 1 and FIND 3 PCR-fragments (FIGS. 7 A and B) weremixed in equimolar amounts together with 100 μl of 2×BAL31 buffer and 10μl sterile water to a final volume of 200 μl. A smaller volume of 22.5μl was prepared to be used as an enzymatically untreated blank. Thisconsisted of 4.5 μl FIND 1-fragment and 4.5 μof FIND 3, 11.25 μl 2×BAL31nuclease buffer and 2.25 μl sterile water. 1.5 ml sterile eppendorftubes with DNA and 2×BAL31 nuclease buffer and water as described werepre-incubated in a 30° C. water-bath in a cold-room of +4° C. for 10minutes. Meanwhile five sterile eppendorf tubes were prepared with 4 μleach of a 200 mM solution of EGTA. These were marked 1-9 minutes. In thesame way a tube with 2.5 μl 200 mM EGTA was prepared for the blankuntreated DNA-solution. The working concentration of EGTA is 20 mM.After the 10 minutes pre-incubation BAL31 Nuclease was added to the tubewith the larger volume to a final concentration of 1 Unit/μg of DNA (10μl of 1 U/μl solution). After t=1, 3, 5, 7 and 9 minutes the tube wasmixed and samples of 36 μl was removed and added to the tubes with 4 μlof EGTA and placed onto ice. At the same time the blank volume of 22.5μl was removed and added to the prepared 2.5 μl of EGTA and also placedon ice. The tubes were then placed in a 65° C. water-bath for heatinactivation of the enzyme and then replaced onto ice.

Purification of Digestion Produced Fragments

The volumes in the tubes were corrected to 100μl each and aphenol/chloroform/isoamylalcohol extraction was performed. 50 μl ofbuffered phenol was added to each tube together with 50 μl of a mixtureof chloroform and isoamylalcohol (24:1). The tubes were vortexed for 30seconds and then centrifuged for 1 minute in a microfuge at 14000 r.p.m.The upper phase was then collected and mixed with 2.5 volumes of 99.5%Ethanol (1/10 was 3M Sodium Acetate, pH 5.2). The DNA was precipitatedfor 1 hour in −80° C. The DNA was then pelleted by centrifugation for 30minutes in a microfuge at 14.000 r.p.m. The pellet was washed once with70% ethanol and then re-dissolved in 10 μl of sterile water.

Analysis of Digestion Produced Purified Fragments on Agarose Gel

5 μl of the dissolved pellet from each time point and from the blankwere mixed with 2.5 μl of loading buffer (25% Ficoll and Bromphenolicblue) and loaded into wells in a 2% agarose gel. The electrophoresis andsubsequent gel extraction of the different timepoints were performed asabove.

Reassembly PCR with BAL31 Nuclease Generated Fragments

The remaining 5 μl of the dissolved pellet from each time point afterphenol-extraction and precipitation were mixed in a PCR-reassemblywithout primers. A portion of 5 μl from the untreated blank was added astemplate to make it possible to generate full length fragments. 40PCR-cycles were run with the PCR-profile and reaction mixture asdescribed above, but without any primers.

PCR with External Primers to Increase the Amount of ReassembledPCR-products

50 μl of the reassembled PCR-product was mixed with PCR reagentsincluding the two external primers as described above to generate a 100μl PCR reaction. This PCR was run for 25 cycles with the profiledescribed above. The amplified PCR-product was analysed on a agarosegel. A band of approximately 1000 bp was visible on the gel after thesecond PCR with the two external primers. The remaining 50 μl from thefirst reassembly PCR, showed only a smear of bands spanning the wholeinterval of the molecular weight marker. The 1000-bp fragment after thesecond PCR was excised and gel-purified as described previously.

Restriction Digestion of Reassembled FIND-fragment and TetracyclineSensitive pBR322 with HindIII and EagI

10 μg of tetracycline deleted pBR322 (10 μl) was cleaved with 2 μl eachof the enzymes HindIII (10 U/μl) and EagI (10 U/μl) (4 U enzyme/μgvector) in a mixture with 10 μl 10×buffer B (supplied with the enzymes)and water to 100 μl. All of the agarose purified reassembledFIND-fragment was cleaved with the same enzymes in a similar 100 μlreaction mixture. The tubes were incubated in a 37° C. water bath for 14hours.

Gel Purification of Restriction Digested Vector and Restriction DigestedReassembled FIND-fragment

The cleavage reactions were mixed were analysed on a 2% agarose gel. Therestriction digested tetracycline-deleted pBR322 showed a cleavageproduct of about 600 bp. This corresponds well with the expected size of635 bp. The band of the cleaved plasmid was cut out and gel-extracted aspreviously described. The reassembled cleaved FIND-product was about1000 bp long and was gel extracted in the same manner as the plasmid.

Spectrophotometer estimations of the restriction digested-plasmid andFIND-fragment gave the following indications of DNA-concentrations:plasmid 13.5 μg/ml; reassembled cleaved FIND-fragment 77.3 μg/ml.

Ligation of Reassembled Restriction Digested FIND-fragment withTetracycline Deleted Restriction Digested pBR322

9.6 μg of purificated cleaved tetracyclineresistance gene-deleted pBR322was ligated to 2.76 μg purified reassembled restriction digestedFIND-fragment at 12° C. water bath for 16 hours. 50 μl of the vector wasmixed with 60 μl of the insert and 15 μl of 10×buffer (supplied with theenzyme) 7.5 μl ligase (5 U/μl) and sterile water to a final volume of150 μl. A ligation of 2 μg restriction digested tetracyclineresistancegene-deleted pBR322 without any insert was also performed in the samemanner.

Transformation of Chemically Competent E coli BMH 71-18 with the LigatedReassembled FIND-insert and pBR322

The ligation reactions were purified by phenol/chloroform extraction asdescribed above. The upper phase from the extraction was collected andmixed with 2.5 volumes of 99.5% Ethanol (1/10 was 3M Sodium Acetate, pH5.2). The DNA was precipitated for 1 hour in −80° C. The DNA was thenpelleted by centrifugation for 30 minutes in a microfuge at 14.000r.p.m. The pellet was washed once with 70% ethanol and then re-dissolvedin 10 μl of sterile water. 5 μl of each ligation was separately mixedwith 95l chemically competent E coli BMH 71-18 incubated on ice for 1hour and then transformed accordingly to the modified protocol of Detlef(Modified Hanahan, revised M. Scott, F. Hochstenbach and D. Güssow1989). After one hour's growth the bacteria from the two transformationswere spread onto ampicillin containing agar plates (100 μg/ml). Theplates were grown upside-down in a 37° C. incubator for 14 hours.

Testing of Ampicillin-resistant Transformant for Tetracycline-resistantRecombinants

The transformation with reassembled FIND-fragment andtetracycline-deleted pBR322 gave 122 ampicillin-resistant transformants.The religated cleaved empty tetracycline-deleted pBR322 gave 100transformants. The transformants from both categories were transferredwith sterile picks one at a time to tetracycline (10 μg/ml) containingagar plates and to ampicillin containing plates at the same time and tocorresponding locations. These plates were incubated in 37° C. incubatorfor 14 hours.

Counting of Tetracycline Resistant Recombinants

The colonies on both the tetracycline plates and the ampicillin plateswere counted the following day for both transformants.

FIND Experiment II

The above described methods were used for a second BAL31 Nucleasetreatment with a mixture of 5 μg of FIND 1 and 5 μg of FIND 3 asdescribed above and in the overview in FIG. 1. This time newPCR-fragments had been generated with the estimated concentrations of192.25 μg/ml for FIND 1 and 231.5 μg/ml for FIND 3. The followingreaction micture was used: 26 μl FIND 1, 21.6 μl FIND 3, 100 μl 2×BAL31exonulease buffer, 9.9 μl BAL31 Nuclease and water to 200 μl. A blankwas also prepared with 13 μl FIND 1 and 10.8 μl FIND 3, 36 μl 2×BAL31exonulease buffer, 0 μl BAL31 Nuclease and water to 72 μl.

The BAL31 digestion was performed as described in the previousexperiment and samples were withdrawn at the same timepoints to tubeswith 200 mM EGTA to get a final concentration of 20 mM EGTA. Theexonuclease in the resulting samples was heat-inactivated as describedabove and the fragments where extracted, precipitated and 50% wereloaded on agarose gel. After the same appearance as previously on thegel had been established, the samples were purified and 2 sequentialPCR-reactions were run as before. The final PCR-fragment was cloned intotetracycline deleted pBR322 under the same conditions as above. Theligation was then electroporated into electrocompetent cells asdescribed (Dower, W. J., J. F. Miller, and C. W. Ragsdale. 1988: Highefficiency transformation of E.coli by high voltage electroporation.Nucleic Acids Res. 16:6127.) and plated on ampicillin agar plates asbefore. Several thousands of transformants were achieved. 397 of thesewere transported as described above to tetracycline agar plates andampicillin agar plates at the same time. The amount of tetracyclinerevertants were counted the following day after incubation in a 37° C.incubator for 14 hours.

The tetracyclin recombinants were then plated for separate colonies ontonew tetracyclin plates. Separate colonies were then inoculated intoliquid cultures with 1×TB-media (Terrific Broth; Molecular cloning; ALABORATORY MANUAL, Second Edition, Cold Spring Harbor Laboratory Press,1989) with 1% Glucose and both ampicillin and tetracycline with theabove concentrations and grown for plasmid-preparations with QiagenPlasmid Midi Kit (Qiagen Inc., Chatsworth, Calif., USA). Glycerol stocksof these overnight cultures were prepared by mixing 500 μl of bacterialculture with 215 μl of 50% Glycerol and storing these mixtures at −80°C.

A bacterial PCR-screening with the two external primers mentioned aboveof 40 of the tetracycline-sensitive colonies was performed to estimatethe frequency of empty religated vector among these transformants. Thiswas done with the PCR-mixture mentioned previously scaled down to 25 μlreactions. These were inoculated with one sensitive bacterial colonyeach and the PCR-profile was as above for 30 cycles. The resultingPCR-fragmnets were analysed on gel as described above.

FIND-experiment I: No. of amp.-resistant FIND- No. of tet-resistantFIND- transformants transformants 122 19 Frequency of recombinants: 16%No. of amp.-resistant relig. No. of tet.-resistant sensitive vectorrelig., Vect. 100  0 Frequency of recombinants: 0% FIND-experiment II:No. of amp.-resistant FIND- No. of tet-resistant FIND- transformantstransformants 397 22

Frequency of recombinants: 5.5%

2 out of 40 bacterially PCR-screened sensitive clones were emptyreligated vector. This would then make up 5% of the total number oftransformants. Therefore, 20 out of 397 is empty vector. This increasedthe number of recombinants to 5.8%.

FIND Experiment III

The FIND procedure is not restricted to the usage on tetracycline genes,but can be applied to any type of genes encoding a protein or proteinmotif. This is exemplified by creating a new repetoir of antibodyfragments with mutations evenly spread over the entire antibody variablegenes after FIND treatment.

Single base pair mutations were introduced into the VL and VH-regions ofthe anti-FITC scFv antibody fragment B11 (Kobayashi et al.,Biotechniques Sep. 23, 1997; (3):500-503) by the use of error prone PCRin accordance with Kuipers et al., (Nucleic Acids Res Aug. 25, 1991;19(16):4558) except for a raise in the MgCl₂ concentration from 2 mM to5 mM. This anti FITC scFv antibody fragment was constructed by the useof overlap extension PCR, and the overlap extension procedure hasprevioulsy been used for the random combination of DNA variation(Söderlind et al. Gene Jul. 28, 1995; 160(2):269-272).

The mutated products were then subjected to controlled degradation withBAL31 exonuclease which can be used for removing nucleotides from thetermini of double stranded DNA in a controlled manner. It ispredominantly a 3′ exonuclease (Sambrook et al., Sambrook, J., FritschE. F. and Mantiatis T. Molecular Cloning—a laboratory Manual Cold SpringHarbor Laboratory Press, 2nd edition, 1989) and removes mono nucleotidesfrom both 3′ termini of the two strands of linear DNA. In addition, italso acts as an endonuclease degrading the ss DNA generated by theexonuclease activity. Degradation is completely dependent on thepresence of calcium and the reaction can be stopped at different stagesby adding the calcium chelating agent EGTA. Bal31 works asynchronouslyon a pool of DNA molecules, generating a population of DNA of differentsizes whose termini have been digested to various extents and whosesingle stranded DNA tails vary in length. DNA of interest is digestedwith BAL31 and samples are withdrawn at different times and placed in asolution with EGTA, which does not interfere with the activity of Taqpolymerase. Thus, PCR based reassembly is possible directly after thedigestion procedure. The average length of single-stranded tails createdby digestion of linear ds DNA is dependent both on time of Bal31treatment and the enzyme concentration. High enzyme concentrations of2-5 U/ml yields an average of 5 nucleotides of ssDNA per terminus,whereas 0.1-0.2 U/ml can yield longer ssDNA.

After the treatment of BAL31, the pool of generated DNA fragments ofvarying sizes, which were reassembled as previously described into fulllength scFv genes. The resulting genes were cloned into the phagemidvector pEXmide5 and the resulting library size after transformation was5.7×10⁴ cfu/ug DNA.

Single clones from the library were sequenced to estimate the geneticvariability in the library. The frequencies of mutations found,distributed over the 782 bp long VL-VH-region of the scFv antibodyranged from 1-56 (Table 1). This is a mutation rate ranging from 0.13%to 7.16%, whereas the mutation rate for error prone PCR has beenreported to be 0.7% (Kuipers et al., Nucleic Acids Res Aug. 25, 1991;19(16):4558). This result demonstrates the effect of recombiningmutations in a set of genes, resulting in a varied gene population whichcan be used in selections/ screening of proteins with new and alteredfunctions.

Reagents

AmpliTaq® polymerase was purchased from Perkin-Elmer Corp., dNTPs fromBoehringer Mannheim Biochemica (Mannheim, Germany), and BAL 31 Nucleasefrom New England Biolabs Inc. (Beverly, USA). All restriction enzymeswere purchased from Boehringer Mannheim Biochemica (Mannheim, Germany).Ethidium bromide was purchased from Bio-Rad Laboratories (Bio-RadLaboratories, Hercules, Calif., USA). T4 DNA Ligase was purchased fromBoehringer Mannheim Biochemica (Mannheim, Germany).

Primers

All primers were designed and synthesised at the department with aApplied Biosystems 391 DNA-synthesiser. The restriction sites introducedin each primer are underlined.

Reamplification Primers

For error prone PCR and reamplification PCR after

Bal31 Treatment

3′-primer DL:FITC-b11-VL3′-FLAG-SAL 1:

5′-CAA CTT TCT TGT CGA CTT TAT CAT CAT CAT CTT TAT AAT CAC CTA GGA CCGTCA GCT TGGT-3′ (SEQ ID #10)

5′-primer DL:FITC B11-VH-5′ Nco1:

5′-ACT CGC GGC CCA ACC GGC CAT GGC CGA GGT GCA GCT GTT GGA G-3′ (SEQ ID#11)

Sequencing Primers

Sequencing reversed pEXmide 4: 5′-GGA GAG CCA CCG CCA CCC TAA C-3′ (SEQID #12)

pUC/M 13 reversed primer: 5′-TCA CAC AGG AAA CAG CTA TGA C-3′ (SEQ ID#13)

Plasmids

pEXmide V: 4055 bp NcoI- and SalI-sites are marked with underline textis shown in FIG. 8.

Error Prone PCR

The error prone PCR reactions were carried out in a 10×buffer containing500 mM NaCl, 100 mM Tris-HCl, pH 8.8, 5 mM MgCl₂ 100 μg gelatine(according to Kuipers et al Nucleic Acids Res. Aug. 25, 1991; 19(16):4558) except for a raise in the MgCl₂ concentration from 2 mM to 5mM).

For each 100 μl reaction the following was mixed:

dATP 5 mM 5 μl

dGTP 5 mM 5 μl

dTTP 10 mM 10 μl

dCTP 10 mM 10 μl

20 μM 3′ primer 1.5 μl

20 μM 5′-primer 1.5 μl

10×Kuipers buffer 10 μl

sterile mp H₂O 46.3 μl

The template scFv FITC B11 in pEXmideV vector (24.5 ng/μl) was added atan amount of 42 ng. 10 μl of 10 mM MnCl₂ was added and the tube waschecked that no precipitation of MnO2 occurred. At last 5 Units of Taqenzyme was added. The error prone PCR was run at the followingtemperatures for 25 cycles without a hot start: 94° C. 1′, 45° C. 1′,72° C. 1′, using a 1 second ramp time, followed by a rapid cooling to 4°C. The resulting product was an error proned insert over the scFv FITCof 782 bp. This insert was purified with Qiaqucik PCR purification kit,before BAL 31 Nuclease treatment.

BAL31 Treatment

Error proned purified insert of the FITC B11 was digested with 0.5 U BAL31 enzyme/pg insert DNA. 1.5 ml sterile eppendorf tubes with DNA,2×BAL31 Nuclease buffer and water were pre-incubated in 30° C. for 10minutes. After this pre-incubation, BAL31 Nuclease was added except forone control tube to a final concentration of 0.5 Unit/pg of DNA. Thecontrol tube, thus, contained only DNA buffer and water. After t=2′, 4′,6′, 8′ and finally 10 minutes, the tube was mixed and samples wereremoved and added to the tubes with EGTA and placed on ice. The workingconcentration of EGTA was 20 mM. At the same time the control volume wasremoved from the water bath and this sample was also mixed with EGTA andplaced on ice. The tubes were then placed in a 65° C. water-bath forheat inactivation of the enzyme and then replaced onto ice.

Reassembly of BAL31 Generated Fragments

The reassembly of the generated fragment pools were performed aspreviously described in two subsequent PCR reactions. The first PCRreaction was performed without the addition of any external primers bymixing equal amounts of the different time pools in a standard PCRreaction. The PCR reaction was run at 40 cycles consisting of followingprofile: denaturation (94° C. for 1 minute), primer annealing (55° C.for 1 minute) and extension (72° C. for 1 minute) using a 1 second ramptime. The PCR reactions contained, unless otherwise noted, 5 μl of eachprimer (20 μM), 16 μl of a dNTP mixture (1.25 mM each of dTTP, dATP,dCTP and dGTP), 10 μl 10×reaction buffer supplied with the enzyme, 0.5μl AmpliTaq® thermostable DNA polymerase (5 U/μl) (Perkin-Elmer Corp.)and water to a final volume of 100 μl.

The reassembled products were then reamplified with a PCR containing the3′- and 5′-external primers to generate an insert of the correct sizeand thereby also introducing the restriction sites NcoI and SalI forcloning into the pEXmideV vector. The PCR reaction was run at 25 cyclesconsisting of following profile: denaturation (94° C. for 1 minute),primer annealing (55° C. for 1 minute) and extension (72° C. for 1minute) using a 1 second ramp time. The PCR reactions contained, 5 μl ofeach primer (20 μM), 16 μl of a dNTP mixture (1.25 mM each of dTTP,dATP, dCTP and dGTP), 10 μl 10×reaction buffer supplied with the enzyme,0.5 μl AmpliTaq® thermostable DNA polymerase (5 U/μl) (Perkin-ElmerCorp.) and water to a final volume of 100 μl. The subsequent insert waspurified on a 2% agarose gel using the Qiaquick gel extraction kit(Kobayashi et al., Biotechniques Sep. 23, 1997; (3):500-503).

Cloning in the PEXMIDEV Phagmid Vector

The insert and vector were digested with the NcoI and SalI enzymes fromBoehringer Mannheim. The insert was cleaved with 10 U enzyme/μg DNA andvector with 4 U/μg DNA. The insert was then gel purified as describedpreviously and the vector was purified using the Microcon 100 microconcentrators (Amicon, Inc., Beverly, Mass. 01915, USA). The vector wasthen cleaved with a third enzyme, the Pst I enzyme, who's restrictionsite is located in between the first two enzymes. The vector was gelpurified with the Qiaquick gel extraction kit (Qiagen GmbH, Hilden,Germany). Insert and purified vector were ligated with 25 U T4 DNAligase/ug DNA (Boehringer Mannheim) at a vector to insert ratio of 590ng vector to 240 ng insert (12:1 molar ratio) for 14 hours at 12° C. Theligation reactions were purified by phenol chloroform extraction andethanol precipitation and subsequently transformed into electrocompetent Top 10 F′ bacterial cells. The library size was determined to5.7×10⁴ cfu/ug DNA. Glycerol stocks were produced after transformationaccording to J. Engberg et al (Molecular Biotechnology Vol 6, 1996p287-310) and stored at −20° C.

Sequencing

Separate colonies from the glycerol stock library were grown and plasmidpreparations were performed with Promega Wizard Plus Minipreps DNApurification System (Promega, Madison, Wis. USA). The VL and VH insertof these plasmids were amplified with a PCR containing the 3′- and5′-external primers to generate an insert of the correct size. Theseinserts were then sequenced with Big Dye Dyedeoxy™ Terminator CycleSequencing Kit. The sequencing was performed on a ABI Prism 377 DNASequencer.

TABLE 1 Number of mutations in the 782 bp long scFv sequences after FINDtreatment Clone Number of Mutations 1  1 2  5 3  8 4 23 5 50 6 56 7 10 826 9 38 10  18

13 1 37 DNA Artificial Sequence Primer 1 cagcttatca tcgataagctttaatgcggt agtttat 37 2 38 DNA Artificial Sequence Primer 2 cgtagcccagcgcgtcggcc gccatgccgg cgataatg 38 3 59 DNA Artificial Sequence Primer 3cactatggcg tgctgctagc gctatatgcg ttgatgcaat ttctatgagc acccgttct 59 4 65DNA Artificial Sequence Primer 4 tctcaagggc atcggtcgac gctctcccttatgcgactcc tgcattagga atcagcccag 60 tagta 65 5 710 DNA ArtificialSequence Theoretical insert 5 ctagcgctat atgcgttgat gcaatttctatgagcacccg ttctcggagc actgtccgac 60 cgctttggcc gccgcccagt cctgctcgcttcgctacttg gagccactat cgactacgcg 120 atcatggcga ccacacccgt cctgtggatcctctacgccg gacgcatcgt ggccggcatc 180 accggcgcca caggtgcggt tgctggcgcctatatcgccg acatcaccga tggggaagat 240 cgggctcgcc acttcgggct catgagcgcttgtttcggcg tgggtatggt ggcaggcccc 300 gtggccgggg gactgttggg cgccatctccttgcatgcac cattccttgc ggcggcggtg 360 ctcaacggcc tcaacctact actgggctgcttcctaatgc aggagtcgca taagggagag 420 cgtcgaccga tgcccttgag agccttcaacccagtcagct ccttccggtg ggcgcggggc 480 atgactatcg tcgccgcact tatgactgtcttctttatca tgcaactcgt aggacaggtg 540 ccggcagcgc tctgggtcat tttcggcgaggaccgctttc gctggagcgc gacgatgatc 600 ggcctgtcgc ttgcggtatt cggaatcttgcacgccctcg ctcaagcctt cgtcactggt 660 cccgccacca aacgtttcgg cgagaagcaggccattatcg ccggcatggc 710 6 322 DNA Artificial Sequence Theoreticalinsert 6 gagccactat cgactacgcg atcatggcga ccacacccgt cctgtggatcctctacgccg 60 gacgcatcgt ggccggcatc accggcgcca caggtgcggt tgctggcgcctatatcgccg 120 acatcaccga tggggaagat cgggctcgcc acttcgggct catgagcgcttgtttcggcg 180 tgggtatggt ggcaggcccc gtggccgggg gactgttggg cgccatctccttgcatgcac 240 cattccttgc ggcggcggtg ctcaacggcc tcaacctact actgggctgattcctaatgc 300 aggagtcgca taagggagag cg 322 7 645 DNA ArtificialSequence Experimentally determined sequence 7 ccgttnaagn nnacacagttanattgttaa ngcagtcagg caccgtgtat gaaatctaac 60 aatgcgctca tcgtcatcctcggnaccgtc accctggatg ttgtaggcat aggcttggtt 120 atgccggtac tgccgggcctcttgcgggat atcgtccatt ccgacagnat cgccagtcac 180 tatggngtgc tgctagcgctatatgcgttg atgcaatttc tatgagcacc cgttctcgga 240 gcactgtccg accgctttggccgccgccca gtcctgctcg cttcgctact tggagccact 300 atcgactacg cgatcatggcgaccacaccc gtcctgtgga tcctctacgc cggacgaatc 360 gatggccgga atcaccggggtcacaggtgc ggntgctggn gcctatttcg ccgacatcaa 420 cgatggggaa agatcnggctcgncactncg ggctcatnag nntttggttt cggcntgggt 480 attggtngga agncccccanggccgggggg attgttngng ngccaacttc cttggattga 540 acaatnccct nggggggggggggttcancn ggcncaacct attnntggga ttnttncnna 600 tnnagagtcg ataaggaggngnnggccant ccntgnagcc caccc 645 8 716 DNA Artificial SequenceExperimentally determined sequence 8 cagtatgacc atnnnctagc ttctcgncgagacgtttggt ngcnggacca gttacgaagg 60 cttgagcnag ggagttgaag attccntatactnaatgnga taggnctatc atcggngggc 120 tccanagata gcggncancg ncnacanatgacccagagct ntgccggcan cagtcctacg 180 agtngnatga tnaagtagan aggcataattggggngacga tagtcatgnc ccgcggccac 240 cggaaggagc ttaatgggtt gnnggctctcaagggcatcg gtcgacgctc tcccttatgt 300 gactcntgna ttaggaatca gcccagttngctaggtttgn ggccgnttgn aancaacccc 360 cgnccnnana gggaattgnt gnaatnnaaagggngtttgg gngncccaac aagtcccccc 420 cgngcnanng ggggccctcc caccaattnccccacggccg aaaaaaaang ttttcaatna 480 agccccnagg tnggggaacc cctnttcttcccccatcggn gganatttgg ntgaattttt 540 ggggnccaan anncccnnct ttngggtccgntnttatntc ccncccacaa ttnnttcccg 600 tttnggggnn nnntccnaan gaaggttttntttccccccc natttccnct ttatncnntt 660 tntnntttnn nnatagaaaa anaaaantttgggggngcca aggtttnata atattt 716 9 4054 DNA Artificial Sequence pEXmideV 9 aagcttgcat gcaaattcta tttcaaggag acagtcataa tgaaatacct attgcctacg 60gcagccgctg gattgttatt actcgcggcc caaccggcca tggcatgagc ggccgcccgg 120gcggcgcgcc ctgcaggcta gcactagtgg taccgtcgac aagaaagttg agcccaaatc 180ttcaactaag acgcacacat caggaggtta gggtggcggt ggctctccat tcgtttgtga 240atatcaaggc caatcgtctg acctgcctca acctcctgtc aatgctggcg gcggctctgg 300tggtggttct ggtggcggct ctgagggtgg tggctctgag ggtggcggtt ctgagggtgg 360cggctctgag ggaggcggtt ccggtggtgg ctctggttcc ggtgattttg attatgaaaa 420gatggcaaac gctaataagg gggctatgac cgaaaatgcc gatgaaaacg cgctacagtc 480tgacgctaaa ggcaaacttg attctgtcgc tactgattac ggtgctgcta tcgatggttt 540cattggtgac gtttccggcc ttgctaatgg taatggtgct actggtgatt ttgctggctc 600taattcccaa atggctcaag tcggtgacgg tgataattca cctttaatga ataatttccg 660tcaatattta ccttccctcc ctcaatcggt tgaatgtcgc ccttttgtct ttagcgctgg 720taaaccatat gaattttcta ttgattgtga caaaataaac ttattccgtg gtgtctttgc 780gtttctttta tatgttgcca cctttatgta tgtattttct acgtttgcta acatactgcg 840taataaggag tcttaataag ggagcttgca tgcaaattct atttcaagga gacagtcata 900atgaaatacc tattgcctac ggcagccgct ggattgttat tactgaattc actggccgtc 960gttttacaac gtcgtgactg ggaaaaccct ggcgttaccc aacttaatcg ccttgcagca 1020catccccctt tcgccagctg gcgtaatagc gaagaggccc gcaccgatcg cccttcccaa 1080cagttgcgca gcctgaatgg cgaatggcgc ctgatgcggt attttctcct tacgcatctg 1140tgcggtattt cacaccgcat acgtcaaagc aaccatagta cgcgccctgt agcggcgcat 1200taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc agcgccctag 1260cgcccgctcc tttcgctttc ttcccttcct ttctcgccac gttcgccggc tttccccgtc 1320aagctctaaa tcgggggctc cctttagggt tccgatttag tgctttacgg cacctcgacc 1380ccaaaaaact tgatttgggt gatggttcac gtagtgggcc atcgccctga tagacggttt 1440ttcgcccttt gacgttggag tccacgttct ttaatagtgg actcttgttc caaactggaa 1500caacactcaa ccctatctcg ggctattctt ttgatttata agggattttg ccgatttcgg 1560cctattggtt aaaaaatgag ctgatttaac aaaaatttaa cgcgaatttt aacaaaatat 1620taacgtttac aattttatgg tgcactctca gtacaatctg ctctgatgcc gcatagttaa 1680gccagccccg acacccgcca acacccgctg acgcgccctg acgggcttgt ctgctcccgg 1740catccgctta cagacaagct gtgaccgtct ccgggagctg catgtgtcag aggttttcac 1800cgtcatcacc gaaacgcgcg agacgaaagg gcctcgtgat acgcctattt ttataggtta 1860atgtcatgat aataatggtt tcttagacgt caggtggcac ttttcgggga aatgtgcgcg 1920gaacccctat ttgtttattt ttctaaatac attcaaatat gtatccgctc atgagacaat 1980aaccctgata aatgcttcaa taatattgaa aaaggaagag tatgagtatt caacatttcc 2040gtgtcgccct tattcctttt ttgcggcatt ttgccttcct gtttttgctc acccagaaac 2100gctggtgaaa gtaaaagatg ctgaagatca gttgggtgca cgagtgggtt acatcgaact 2160ggatctcaac agcggtaaga tccttgagag ttttcgcccc gaagaacgtt ttccaatgat 2220gagcactttt aaagttctgc tatgtggcgc ggtattatcc cgtattgacg ccgggcaaga 2280gcaactcggt cgccgcatac actattctca gaatgacttg gttgagtact caccagtcac 2340agaaaagcat cttacggatg gcatgacagt aagagaatta tgcagtgctg ccataaccat 2400gagtgataac actgcggcca acttacttct gacaacgatc ggaggaccga aggagctaac 2460cgcttttttg cacaacatgg gggatcatgt aactcgcctt gatcgttggg aaccggagct 2520gaatgaagcc ataccaaacg acgagcgtga caccacgatg cctgtagcaa tggcaacaac 2580gttgcgcaaa ctattaactg gcgaactact tactctagct tcccggcaac aattaataga 2640ctggatggag gcggataaag ttgcaggacc acttctgcgc tcggcccttc cggctggctg 2700gtttattgct gataaatctg gagccggtga gcgtgggtct cgcggtatca ttgcagcact 2760ggggccagat ggtaagccct cccgtatcgt agttatctac acgacgggga gtcaggcaac 2820tatggatgaa cgaaatagac agatcgctga gataggtgcc tcactgatta agcattggta 2880actgtcagac caagtttact catatatact ttagattgat ttaaaacttc atttttaatt 2940taaaaggatc taggtgaaga tcctttttga taatctcatg accaaaatcc cttaacgtga 3000gttttcgttc cactgagcgt cagaccccgt agaaaagatc aaaggatctt cttgagatcc 3060tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt 3120ttgtttgccg gatcaagagc taccaactct ttttccgaag gtaactggct tcagcagagc 3180gcagatacca aatactgtcc ttctagtgta gccgtagtta ggccaccact tcaagaactc 3240tgtagcaccg cctacatacc tcgctctgct aatcctgtta ccagtggctg ctgccagtgg 3300cgataagtcg tgtcttaccg ggttggactc aagacgatag ttaccggata aggcgcagcg 3360gtcgggctga acggggggtt cgtgcacaca gcccagcttg gagcgaacga cctacaccga 3420actgagatac ctacagcgtg agctatgaga aagcgccacg cttcccgaag ggagaaaggc 3480ggacaggtat ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg agcttccagg 3540gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg 3600atttttgtga tgctcgtcag gggggcggag cctatggaaa aacgccagca acgcggcctt 3660tttacggttc ctggcctttt gctggccttt tgctcacatg ttctttcctg cgttatcccc 3720tgattctgtg gataaccgta ttaccgcctt tgagtgagct gataccgctc gccgcagccg 3780aacgaccgag cgcagcgagt cagtgagcga ggaagcggaa gagcgcccaa tacgcaaacc 3840gcctctcccc gcgcgttggc cgattcatta atgcagctgg cacgacaggt ttcccgactg 3900gaaagcgggc agtgagcgca acgcaattaa tgtgagttag ctcactcatt aggcacccca 3960ggctttacac tttatgcttc cggctcgtat gttgtgtgga attgtgagcg gataacaatt 4020tcacacagga aacagctatg accatgatta cgcc 4054 10 61 DNA Artificial SequencePrimer 10 caactttctt gtcgacttta tcatcatcat ctttataatc acctaggaccgtcagcttgg 60 t 61 11 43 DNA Artificial Sequence Primer 11 actcgcggcccaaccggcca tggccgaggt gcagctgttg gag 43 12 22 DNA Artificial SequencePrimer 12 ggagagccac cgccacccta ac 22 13 22 DNA Artificial SequencePrimer 13 tcacacagga aacagctatg ac 22

What is claimed is:
 1. A method for generating at least onepolynucleotide sequence from a parent polynucleotide sequence encodingat least one protein motif, comprising the steps of a) digesting theparent polynucleotide sequence with an exonuclease to generate apopulation of fragments; b) contacting said fragments with an undigestedtemplate polynucleotide sequence under annealing conditions; c)amplifying the fragments that anneal to the template in step b) togenerate at least one polynucleotide sequence encoding at least oneprotein motif having altered characteristics as compared to the at leastone protein motif encoded by said parent polynucleotide.
 2. A methodaccording to claim 1 wherein the parent polynucleotide isdouble-stranded and the method further comprises the step of generatingsingle-stranded polynucleotide sequence from said double-strandedfragments prior to step b).
 3. A method according to claim 1 whereintemplate polynucleotide sequence is the parent polynucleotide sequence.4. A method for generating at least one polynucleotide sequence from aparent polynucleotide sequence encoding at least one protein motif, inwhich said polynucleotide sequence has altered sequence at its terminias compared to said parent polynucleotide sequence comprising the stepsof (a) digesting said parent polynucleotide sequence with an exonucleaseto generate a population of fragments; (b) contacting said fragmentswith an undigested template polynucleotide sequence which is a variantof said parent polynucleotide sequence, under annealing conditions; (c)amplifying the fragments that anneal to the template in step b) togenerate at least one polynucleotide sequence encoding at least oneprotein motif encoded by said parent polynucleotide.
 5. A method forgenerating at least one polynucleotide sequence from a parentpolynucleotide sequence encoding at least one protein motif, in whichsaid polynucleotide sequence has altered sequence at its center ascompared to said parent polynucleotide sequence comprising the steps of(a) digesting a population of variant polynucleotide sequences with anexonuclease to generate a population of fragments; (b) contacting saidfragments with said parent polynucleotide sequence under annealingconditions, said parent polynucleotide sequence being undigested; (c)amplifying the fragments that anneal to the parent in step b) togenerate at least one polynucleotide sequence encoding at least oneprotein motif encoded by said parent polynucleotide.
 6. A methodaccording to claim 4 wherein the exonuclease is BAL31.
 7. A methodaccording to claim 4 wherein the parent polynucleotide sequence encodesan antibody or fragment thereof.
 8. A method according to claim 1wherein the parent polynucleotide sequence encodes an enzyme.
 9. Amethod according to claim 4 further comprising the step of screening theat least one polynucleotide generated in step c) for desiredcharacteristics.
 10. A method according to claim 4 further comprisingthe step of expressing the at least one polynucleotide generated in stepc) and screening the resulting polypeptide for desired characteristics.11. A method for preparing a pharmaceutical composition which comprises,following the identification of a polynucleotide with desiredcharacteristics by a method according to claim 1, adding saidpolynucleotide to a pharmaceutically acceptable carrier.
 12. A methodfor preparing a pharmaceutical composition which comprises, followingthe identification of a polypeptide with desired characteristics by amethod according to claim 8, adding said polypeptide to apharmaceutically acceptable carrier.
 13. A process which comprises,following the identification of a polynucleotide by a method of claim 1,the manufacture of that polynucleotide, in whole or in part, optionallyin conjunction with additional polynucleotide sequence.
 14. A processwhich comprises, following the identification of a polypeptide by amethod according to claim 9, the manufacture of that polypeptide, inwhole or in part, optionally in conjunction with additionalpolypeptides.
 15. A process according to claim 14 wherein thepolypeptide is an antibody or fragment thereof.
 16. A process accordingto claim 14 wherein the polypeptide is an enzyme.
 17. A method accordingto claim 5 further comprising the step of screening the at least onepolynucleotide generated in the step c) for desired characteristics. 18.A method according to claim 5 further comprising the step of expressingthe at least one polynucleotide generated in step c) and screening theresulting polypeptide for desired characteristics.